Glycoproteins are glycosylated polypeptides. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of sugars, e.g., N-acetylgalactosamine, galactose, or xylose to a hydroxylamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The N-linked oligosaccharides are further differentiated into 3 subgroups, these being the high mannose type, the complex type, and the hybrid type. N-linked oligosaccharides are frequently branched, where branching commonly occurs either at a mannose residue or at an N-acetylglucosamine residue. These branched structures are called biantennary, if there are two branches, and triantennary if there are three branches.
Existing methods for analyzing carbohydrate structure rely on complex multi-step procedures. These procedures involve techniques such as mass spectrometry, NMR, fast atom bombardment, complex chromatography techniques (high pressure liquid chromatography, gas phase chromatography, ion-exchange and reverse-phase chromatography) and complex series of chemical reactions (methylation analysis, periodate oxidation, and various hydrolysis reactions) and have all been used in various combinations to determine the sequence of oligosaccharides and the features of their glycosidic linkage. Each method can provide certain pieces of information about carbohydrate structure, but each has disadvantages. For example, fast atom bombardment (Dell, A., Adv. Carbohydr. Chem. Biochem. 45 (1987) 19-72) can provide some size and sequence data but does not provide information on linkage positions or anomeric configuration. NMR is the most powerful tool for analyzing carbohydrates (Vliegenthart et al., Adv. Carbohydr. Chem. 41 (1983) 209-375) but is relatively insensitive and requires large quantities of analyte. These methods have been reviewed by Spellman, M. W., Anal. Chem. 62 (1990) 1714-1722; Lee, K. B., et al., Appl. Biochem. Biotechnol. 23 (1990) 53-80; and Geisow, M. J., Bio/technology 10 (1992) 277-280.
Removal of carbohydrate moieties from a purified glycosylated protein may be accomplished chemically or enzymatically. For instance, chemical deglycosylation by exposing the polypeptide to the compound trifluoromethanesulfonic acid or an equivalent compound can result in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Sojar, H. T., and Bahl, O. P., Arch. Biochem. Biophys. 259 (1987) 52-57, and by Edge, A. S. B., et al., Anal. Biochem. 118 (1981) 131-137. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura, N. R., and Bahl, O. P., Meth. Enzymol. 138 (1987) 350-359.
There are glycosylated enzymes known to the art where the carbohydrate moiety is required for maintaining enzymatic activity. An example therefor has been described by Barbaric, S., et al., Arch. Biochem. Biophys. 234 (1984) 567-575. Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was deglycosylated by endo-β-N-acetylglucosaminidase H or by HF treatment. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure.
Houba, H. J. et al. Bioconjugate Chem. 7 (1996) 606-611 describe the modification of human beta-glucuronidase using NaIO4 and NaBH4, to improve the retention of the enzyme in the circulation. The modified enzyme was used to prepare immunoconjugates.
Expression of heterologous proteins in yeast often results in heavily glycosylated proteins with a high mannose content (Tanner, W., and Lehle, L., Biochim Biophys Acta 906 (1987) 81-99). One example therefor is alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar) which was produced as a heterologous protein in the methylotrophic yeast Hansenula polymorpha (Fellinger, A. J., et al., Yeast 7 (1991) 463-473). In C. tetragonoloba the alpha-galactosidase is a glycoprotein. The alpha-galactosidase secreted by H. polymorpha was also glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U/mg compared to 100 U/mg for the native guar alpha-galactosidase. Notably, deglycosylation of the alpha-galactosidase restored the specific activity completely.
Purifying from native mammalian host tissue a protein to be used for forming a conjugate bears the risk that an unwanted compound such as an inhibitor or a pathogen may copurify. E.g., bovine alkaline phosphatase isolated from bovine tissue may be contaminated with pathogenic bovine prion protein. For this reason, recombinant expression of the desired protein in a microbial host such as yeast is preferred. Very much preferred is a methylotrophic yeast as a microbial host. Expression of a desired protein in yeast can take advantage of intracellular trafficking pathways such as the secretory pathway which includes modification of the desired protein by glycosylation.
EP 1,176,205 discloses highly active eukaryotic alkaline phosphatase expressed as a heterologous protein in Pichia pastoris which is also glycosylated by the yeast when targeted to the secretory pathway. Notably, the properties of the yeast-derived enzyme are similar to those of the native glycosylated enzyme purified from bovine intestine.
Accordingly, the specific activity of the alkaline phosphatase expressed as a heterologous protein in Pichia pastoris is reported to have a specific activity of 7,000 U/mg. Thus, the yeast-specific carbohydrate moiety does not interfere with the enzymatic activity of the free enzyme.
Alkaline phosphatase is an example of an enzyme that is used frequently as a label in analytical methods for the detection of chemical or biological substances. Most of these methods rely on what are known as “specific binding” reactions in which a substance to be detected, referred to as a “target molecule” or “analyte”, reacts specifically and preferentially with a corresponding “molecule capable of binding to a target molecule” or “receptor”. Most well-known specific binding reactions occur between immunoreactants, e.g., antibodies, and antigens or haptens. By “hapten” is meant any molecule which can act as an antigen but which is incapable by itself of eliciting an immune response. In order to elicit an appropriate antibody response, a hapten can be bound, typically via covalent attachment, to an immunogenic carrier to produce an immunogenic conjugate capable of eliciting antibodies specific for the hapten.
Also known are other specific binding reactions, such as avidin or streptavidin with biotin, a carbohydrate with a lectin, or a hormone with a hormone receptor. In addition, the term “specific binding” also includes the interaction of complementary nucleic acids or analogs thereof in a hybridization reaction. Moreover, the term “specific binding” is known to occur between a protein and a nucleic acid or a nucleic acid analog. An example of a nucleic acid analog is a phosphorothioate or a peptide nucleic acid (“PNA”).
Since samples to be analyzed contain the target molecules often in very small amounts, methods based on immunoassays are preferably used for their detection with which the target molecules can be determined very specifically and exactly. There are many variants of these methods. The various immunological methods of determination may be classified into homogeneous and heterogeneous methods. A solid phase reaction always forms part of the “heterogeneous” method in order to immobilize complexes which contain the substance to be detected and a labelled component, and thus to separate them from unbound labelled components. In the “homogeneous” method variant there is no separation of bound label and unbound label so that bound and unbound label have to be differentiated by other methods.
The “label” is any molecule that produces or can be induced to produce a signal. There are many different “labelled components” known for immunoassays. One part of a labelled component, the label, is an enzyme that needs one or several additional components to produce a signal, and the signal producing system would then include all the components required to produce a measurable signal. Thus, the signal is detected and/or measured by detecting the activity of the enzyme. The additional components may include substrates, coenzymes, enhancers, additional enzymes, substances that react with products that are generated by enzymatic activity, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances. A detailed discussion of suitable signal producing systems can be found in U.S. Pat. No. 4,275,149 and U.S. Pat. No. 5,185,243. Examples of enzymes and substrates include, for example: (a) horseradish peroxidase with hydrogen peroxide as a substrate, wherein the hydrogen peroxide oxidizes a dye precursor, e.g., orthophenylene diamine or 3,3′,5,5′-tetramethyl benzidine hydrochloride; (b) alkaline phosphatase with para-nitrophenyl phosphate as chromogenic substrate; and (c) β-D-galactosidase with a chromogenic substrate, e.g., o-nitrophenyl-β-D-galactopyranoside or with a fluorogenic substrate 4-methylumbelliferyl-β-D-galactopyranoside. However, numerous other enzyme-substrate combinations are known to a person skilled in the art.
Another part of a labelled component is a molecule capable of binding to a target molecule, exemplified by an antibody or a functional fragment of an antibody. Single chain antibodies and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising portions derived from different species, are also encompassed by the term “antibody” as used herein. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the foregoing antibodies retain at least one binding function of the full-length antibody from which they are derived. Preferred functional fragments retain an antigen-binding function of a corresponding full-length antibody. Other examples of a molecule capable of binding to a target molecule in a labelled component are avidin, streptavidin, lectins, nucleic acids or analogs thereof.
A labelled component that comprises two portions, that is to say an enzyme and a molecule capable of binding to a target molecule, can be obtained by forming a “conjugate”, i.e. by conjugating the two portions. A conjugate is a molecule comprised of two or more molecules attached to one another, optionally through a linking group, to form a single structure. The binding can be made either by a direct connection between the molecules or by means of a linking group. An overview on the formation of conjugates, particularly the conjugation of enzymes can be found in Hermanson, G. T., In: Bioconjugate Techniques, Ch. 16, Academic Press, 1996, pp. 630-638. Techniques for conjugating enzymes to proteins are described in O'Sullivan, M. J., and Marks, V., Methods Enzymol. 73 (1981) 147-166.
In a conjugate, the function, that is to say, the activity of the enzyme which is comprised therein as a label can be impaired due to several reasons. For example, in the conjugate the enzyme may have an altered and suboptimal conformation. Another example is an interaction of the enzyme with a molecule with which it forms the conjugate, e.g. an antibody. In such a case impaired enzyme activity in the conjugate could result from steric effects that reduce, e.g., the access of a substrate to the catalytic center of the enzyme. Consequently, an assay for detecting the presence or determining the quantity of a target molecule such as an immunoassay (a detection assay) has a reduced sensitivity in case the labelled component is a conjugate comprising an enzyme with an impaired activity. Conversely, the sensitivity of a detection assay such as an immunoassay can be increased by removing any obstacles that impair the activity of the enzyme in the conjugate that is used in the assay as the labelled component.